EP3512946A1 - Universelles verfahren zur extraktion von nukleinsäuren aus einer unterschiedlichen population von einer oder mehreren arten von mikroben in einer probe - Google Patents

Universelles verfahren zur extraktion von nukleinsäuren aus einer unterschiedlichen population von einer oder mehreren arten von mikroben in einer probe

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Publication number
EP3512946A1
EP3512946A1 EP17851634.0A EP17851634A EP3512946A1 EP 3512946 A1 EP3512946 A1 EP 3512946A1 EP 17851634 A EP17851634 A EP 17851634A EP 3512946 A1 EP3512946 A1 EP 3512946A1
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EP
European Patent Office
Prior art keywords
sample
microbiome
lysis solution
analysis
probiotic
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EP17851634.0A
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English (en)
French (fr)
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EP3512946A4 (de
Inventor
Suneer JAIN
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Sun Genomics Inc
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Sun Genomics Inc
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Publication of EP3512946A1 publication Critical patent/EP3512946A1/de
Publication of EP3512946A4 publication Critical patent/EP3512946A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer

Definitions

  • the present invention relates generally to genomic analysis and more particularly to a method of extracting and analyzing nucleic acid molecules from a diverse population of microbes in a biological sample.
  • the present invention is directed to a method of extracting nucleic acid molecules from a diverse population of microbes in a biological, environmental, dietary supplement, or other ecological microbial organism heterogeneous populations sample and use of nucleic acid or extracts through processing steps and analysis for the determination of probiotic customization to an individual.
  • Processing steps specific to this invention include, RNA or DNA clean-up, fragmentation, separation, or digestion; library or nucleic acid preparation for downstream applications, such as PCR, qPCR, digital PCR, or sequencing; preprocessing for bioinformatic QC, filtering, alignment, or data segregation; metagenomics or human genomic bioinformatics pipeline for microbial species taxonomic assignment; and other organism alignment, identification, and variant interpretation.
  • the invention provides a method for preparing a sample for analysis.
  • the method includes: a) mixing the sample with a first lysis solution comprising a detergent, e.g., SDS, and a chelator, e.g., EDTA; b) adding a second lysis solution having a lysozyme to the mixture of step a); and c) adding a third lysis solution comprising a chaotropic agent, e.g., urea, lithium acetate, guanidine hydrochloride, and the like, to the mixture of step b).
  • Pre-processing steps may include physical lysis may be used to further optimize nucleic acid yield. Examples of mechanical lysis include sonication, bead mixing, and bead mill homogenization.
  • the invention provides a method of monitoring a probiotic treatment of a subject.
  • the method includes:
  • the invention provides a method comprising calculating a probiotic score from probiotic organisms detected in a gut with or without additional chemistry or genetic tests. [0009] In still another aspect, the invention provides a method comprising calculating a score for a microbiome, the score being used to assess if the microbiome is in dysbiosis, neutral, or stable.
  • the invention further provides a computing system comprising: a memory; and one or more processors coupled to the memory, the one or more processors configured to perform operations to perform a method of the present invention.
  • the invention also provides an automated platform for performing a method of the invention.
  • the invention provides an all-in-one method for extracting nucleic acids from a diverse flora of microbes from a biological, environmental, dietary supplement, or other ecological microbial organism heterogeneous populations sample.
  • the invention may be used in determining composition and relative abundance of microbes, via analyzing their respective nucleic acids, in probiotics and environmental samples. DNA is purified and used downstream for nucleic acid analysis (notably metagenomics analysis where genome of more than one species/subspecies is identified).
  • Figure 1 is a schematic diagram illustrating the presence of high prevalence organisms of a microbiome signature of a human (high protein diet, >50 years old, supplement user).
  • Figure 2A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).
  • Figure 2B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).
  • Figure 2C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).
  • Figure 2D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).
  • Figure 3A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non- vegetarian diet).
  • Figure 3B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non- vegetarian diet).
  • Figure 3C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non- vegetarian diet).
  • Figure 3D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non-vegetarian diet).
  • Figure 4A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).
  • Figure 4B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).
  • Figure 4C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).
  • Figure 4D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).
  • Figure 5 is a schematic diagram illustrating the presence lower prevalent organisms and identification of opportunistic pathogens of a microbiome signature of a human.
  • Figure 6 is a schematic diagram illustrating typical probiotics detected in a microbiome signature of a human.
  • Figure 7 is a schematic diagram illustrating typical probiotics detected in a microbiome signature of a human.
  • Figure 8 is a schematic graphical plat illustrating showing comparison of individual relative abundance to database average for normal population.
  • Figure 9 is a table setting forth organisms identified via the method of the invention from a dietary supplement mixed culture.
  • Figure 10 is a table setting forth the classificatin of unique speicies of various microbes stored in the database of the invention.
  • the present invention provides a universal method for extracting nucleic acid molecules from a diverse population of one or more types of microbes in a sample.
  • the types of microbes include: gram-positive bacteria, gram-positive bacterial spores, gram-negative bacteria, archaea, protozoa, helminths, algae, fungi, fungal spores, viruses, viroids, bacteriophages, and rotifers.
  • the diverse population is a plurality of different microbes of the same type, e.g., gram-positive bacteria.
  • the diverse population is a plurality of different types of microbes, e.g., bacteria (gram-positive bacteria, gram-positive bacterial spores and/or gram-negative), fungi, viruses, and bacteriophages.
  • bacteria gram-positive bacteria, gram-positive bacterial spores and/or gram-negative
  • fungi fungi
  • viruses bacteriophages.
  • the sample comprising the microbes may be a biological sample, environmental sample, an artificially created sample (e.g., a laboratory test or control sample, a sample of a probiotic composition or supplement, etc.), or the like.
  • biological samples include tissue samples, blood samples, plasma samples, cerebrospinal fluid samples, urine samples, fecal samples, samples of material obtained from the digestive tract, biological secretions (e.g., semen, vaginal secretions, breast milk, tears, saliva, etc.), and the like.
  • Solid samples may be liquefied or mixed with a solution, and then genetic material of the microbes present in the liquefied sample, mixture, or solution obtained from the mixture may be extracted in accordance with the present invention.
  • the extracted genetic material may be subjected to further processing and analysis such as purification, amplification, and sequencing.
  • the extracted genetic material is subjected to metagenomics analysis to, for example, identify the one or more types of microbes in the sample from which the genetic material was extracted.
  • full whole genome shotgun sequencing can be performed on prepared extracted nucleic acid material from human fecal samples. Preparations include nucleic acid clean up reactions to remove organic solvents, impurities, salts, phenols, and other process inhibiting contaminants. Additional preparations include nucleic acid library prep from each sample where the gDNA is subject to modifications and/or amplifications to prep the sample for sequencing on a sequencing platform such as massively parallel sequencing by synthesis, nanopore, long read, and/or CMOS electronic, sequencing methods.
  • the inventive method allows the successful extraction of genetic material from one or more different types of microbes present in the same sample by subjecting the microbes to three different compositions in a particular order.
  • the method according to the present invention comprises first lysing any gram-negative bacteria present in the sample, which is followed by digesting the polysaccharide component of the cell walls of any yeast and bacteria present in the sample, and then disrupting any cell walls that are intact after the second step with a chaotropic agent.
  • the first step comprises mixing the sample with a first lysis solution comprising a detergent (e.g., sodium dodecyl sulfate (SDS)) and a chelator (e.g., ethylenediaminetetraacetic acid (EDTA)) to lyse any gram-negative bacteria present in the sample.
  • the first lysis solution may further include one or more buffers (e.g., Tris), one or more mild detergents (e.g., Triton X-100), and/or one or more proteases (e.g., proteinase K).
  • the sample is mixed with a second lysis solution comprising a lysozyme to digest the polysaccharide component of any yeast and bacterial cell walls present in the mixture. Because lysozyme may inhibit the activity of the first lysis solution, it is important that contact of the sample with the second lysis solution occurs after treating the sample with the first lysis solution.
  • a third lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.) is added to the mixture to disrupt any cell walls that are not digested by the second lysis solution.
  • a chaotropic agent e.g., urea, lithium acetate, guanidine hydrochloride, etc.
  • the third lysis solution may include a detergent such as SDS.
  • both the first lysis solution and the third lysis solution comprise SDS at a working concentration of between 1-10% w/v.
  • the mixture is further treated with a fourth lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.) and Proteinase K.
  • a chaotropic agent e.g., urea, lithium acetate, guanidine hydrochloride, etc.
  • Proteinase K Proteinase K
  • the sample may be subjected to a pretreatment step that induces germination of the cell walls of the spores before contact with the first lysis solution.
  • the pretreatment step may comprise mixing the sample with a chemical such as a mild detergent, e.g., Tween-80, to induce germination or cultivating the sample under conditions (e.g., temperature) that induce germination.
  • a chemical such as a mild detergent, e.g., Tween-80
  • the chemical is preferably one that does not inhibit, reduce, or modify the activity or effectiveness of the first, second, and third lysis solutions.
  • the method according to the present invention may further include one or more mechanical treatment steps that cause physical lysis by mechanical methods including sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, and the like.
  • a mechanical treatment step is performed before subjecting the sample to the first lysis solution.
  • the method according to the present invention is capable of extracting nucleic acid molecules from a variety of microbes including yeast (i.e., Saccharomyces spp.), gram- negative bacteria (e.g., Acinetobacter spp.), gram-positive bacteria (e.g., Bifidobacterium spp.), viruses (e.g., Sclerotinia spp.), spores (Bacillus spp.) Helminths (tapeworm Echinococcus spp.), Protozoa (Sarcodina - the ameba, e.g., Entamoeba) and phages (e.g., Lactobacillus phages).
  • yeast i.e., Saccharomyces spp.
  • gram- negative bacteria e.g., Acinetobacter spp.
  • gram-positive bacteria e.g., Bifidobacterium spp.
  • viruses
  • a range of lOmg to 5000mg of sample were added to a sterile 2 milliliters (mL) micro centrifuge tube.
  • Bead beating may optionally be performed by adding 400 microliters ( ⁇ ) of bead pure mixture and vortexing for about 30 seconds at 8000 rpm. If, however, high-molecular weight nucleic acids, e.g., genomic DNA, are desired to be obtained, bead beating is preferably avoided.
  • the sample was subjected to a First Lysis Solution by adding about 400 ⁇ of Digestion Buffer (1% w/v SDS, 25 mM Tris HC1, 2.5 mM EDTA, 1% trident-x 100, pH 8) and about 20 ⁇ L ⁇ of Proteinase K to the sample and gently mixed. The mixture was then incubated for about 30 minutes at 55°C.
  • Digestion Buffer 1% w/v SDS, 25 mM Tris HC1, 2.5 mM EDTA, 1% trident-x 100, pH 8
  • a Second Lysis Solution comprising a glucoside hydrolase ("lysozyme") was added to the mixture obtained from the First Lysis Solution Treatment Step to give a final lysozyme concentration of 1 mg/mL and a pH of about 8.0.
  • glucoside hydrolases may be obtained from a variety of sources including egg whites, tears, or mucus or saliva of various animals. The mixture was then incubated for a period of about 1 to 24 hours at 37°C.
  • a Third Lysis Solution comprising 1M lithium acetate in distilled sterile H20 and 5% w/v SDS was added to obtain about a 1 :5 dilution of the mixture resulting from the Second Lysis Solution Treatment Step.
  • the treated mixture was incubated for 15 minutes at 70°C followed by heat shock at 95°C for one minute and then brought to room temperature by placing in a 22°C water bath.
  • the genetic material extracted from the lysed microbes i.e., the nucleic acid molecules present in the mixture after being subjected to the First, Second, and Third Lysis Solution Treatment Steps were then purified to DNA and RNA purification by splitting the mixture into two microcentifuge tubes.
  • DNA was extracted from one tube by adding about 20 ⁇ ⁇ RNAse A and incubating for 5 minutes at room temperature.
  • the mixture was run through a biopolymer tissue homogenizer column. If bead beating was previously performed, subjecting the mixture to the tissue homogenizer column is preferably avoided.
  • the eluate was then centrifuged at 1000 g for 5 minutes.
  • the supernatant was treated with about 400 ⁇ L ⁇ of DNA Lysis Solution (Guanidine HC1, Tris-EDTA, and 70% EtOH) and about 20 ⁇ of Proteinase K, mixed, and then incubated at 55°C for 10 minutes. Then EtOH at -22°C was added and the mixture was mixed by inverting.
  • the mixture may be subjected to one or more additional DNA extraction and purification methods known in the art.
  • RNA stabilization buffer and bead beating is preferred to ensure release and limited degradation of RNA nucleic acid molecules.
  • the extracted and purified genetic material was prepared for sequencing using Illumina index adaptors and checked for sizing and quantity. Low cycle PCR was performed between 1-20 cycles for any input less then 50ng of DNA, otherwise PCR-Free methods of library prep can be utilized for 50ng of nucleic acid or greater.
  • Gel purification was performed using the Qiagen Gel Purification KitTM (Qiagen, Frederick, MD). Clean PCR products were quantified using the QubitTM 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Samples were combined in equimolar amounts.
  • a range from 1000 or greater reads of sequencing for short insert methods can be used for this method.
  • Large insert methods such as Pac BioTM, NanoporeTM, or other next gene sequencing methods can use ⁇ 1000 sequencing reads.
  • Bioinformatics quality filtering was performed before taxonomy assignment.
  • Quality trimming of raw sequencing files may include removal of sequencing adaptors or indexes; trimming 3' or 5' end of reads based on quality scores (Q20>), basepairs of end, or signal intensity; removal of reads based on quality scores, GC content, or non-aligned basepairs; removal of overlapping reads at set number of base pairs.
  • Alignment of processed sequencing files was done using a custom microbial genome database consisting of sequences from refseqTM, GreengeensTM, HMPTM, NCBITM, PATRICTM, or other public/private data repositories or in-house data sets.
  • This database may be used as full genome alignment scaffold, k-mer fragment alignment, or other schemes practiced in the art of metagenomics and bioinformatics.
  • Based off the number of sequencing reads/fragments that match the database genomes we assign a taxonomic identity that is common or unique to the organism.
  • This identifier can be a barcode, nucleotide sequence, or some other computational tag that will associate the matching sequencing read to an organism or strain within a taxonomic group. Some identifiers will be of higher order and would identify domain, kingdom, phylum, class, order, family, or genus of the organism.
  • the present invention is able to identify the organism at the lowest order of strain within a species.
  • the invention includes identification and/or analysis of one or more bacteria contained within our database ( Figure 10).
  • Some selected examples are Bacillus clausii, Bifidobacterium animalis, Pediococcus acidilactici, Acinetobacter indicus, Lactobacillus salivarius, Acinetobacter, Bacillus amyloliquefaciens, Lactobacillus helveticus, Bacillus subtilis, Lactobacillus plantarum, Bifidobacterium longum subsp infantis, Enterococcus hirae, Lactobacillus delbrueckii subsp bulgaricus, Enterococcus, Lactobacillus rhamnosus, Lactococcus lactis, Pseudomonas stutzeri, Lactobacillus acidophilus, Klebsiella and Enterobacter cloacae strain.
  • the invention includes identification and/or analysis of one or more yeast contained within our database ( Figure 10).
  • Figure 10 Some selected examples are Saccharomyces sp. Boulardii, Saccharomyces kudriavzevii, Saccharomyces pastorianus and Saccharomyces cerevisiae.
  • the invention includes identification and/or analysis of one or more phage or viruses contained within our database (figure 10) Some selected examples are Bacillus phage phi29, Enterobacteria phage HK022, Lactobacillus phage A2, Escherichia phage HK639, Phage cdtl, Sclerotinia sclerotiorum partitivirus S segment 2, Burkholderia phage BcepMu, Lactococcus prophage bIL311, Enterococcus phage phiFL4A and Streptococcus phage SMI .
  • the present invention may be used to monitor probiotic treatment in subjects.
  • a sample obtained from the digestive tract of a subject may be obtained and the genetic material of the microbes therein extracted as disclosed herein and subjected to metagenomics analysis.
  • a second sample may be obtained from the digestive tract of the subject and the genetic material of the microbes in the second sample extracted as disclosed herein and subjected to metagenomics analysis, the results of which are compared to the results of the metagenomics analysis of the first sample.
  • the probiotic treatment of the subject may be modified to obtain a desired population of microbes in the gut of the subject.
  • a probiotic that comprises a microbe whose amount is desired to be increased in the gut of the subject may be administered to the subject.
  • the fecal sample may be mixed or cultured for determination of metabolomic of microbial fecal community. Metabolomic profile can then be used to determine probiotic strains that would benefit the individual. Examples of metabolomic profiles include those affecting energy metabolism, nutrient utilization, insulin resistance, adiposity, dyslipidemia, inflammation, short-chain fatty acids, organic acids, cytokines, neurotransmitters chemicals or phenotype and may include other metabolomic markers.
  • the present invention has been successfully used to determine the microbe content of a variety of commercially available probiotics. Additionally, the methods of the present invention are used to determine the microbe content of various probiotics and the microbiome content in the gut of the subject. Based on the microbiome content in the gut of the subject and any desired changes thereto, one may select one or more probiotics that contain the microbes that are desired to be increased and/or maintained in the subject's microbiome health. Where the microbiome represents a full picture of their microbiota and the organisms contained in them from bacteria, fungi, viruses, phages, and parasites.
  • a subject's gut microbiome is determined to contain 25% A and 75% B
  • Probiotic 1 is determined to contain 75% A and 25% B
  • Probiotic 2 is determined to contain 25% A and 75% B. If the subject's gut microbiome is desired to be maintained, one would select Probiotic 2 for administering to the subject. However, if the amounts of A and B in the subject's gut are desired to be 50/50, one may select both Probiotics 1 and 2 to be administered to the subject. Alternatively, one may select Probiotic 1 to be administered to the subject until the amounts of A and B in the subject's gut reaches 50/50.
  • a probiotic formulation e.g., containing equal, varying, or diverse amounts of A and B or other probiotic strains
  • Calculation models utilizing relative abundance of the microbes present in an individual's gut will help determine the type, dose, and cocktail of microbes to include in the probotic. For example, if it is determined that organism A is reduced or absent compared to the general population or previous microbiome analysis, then we would provide probiotic or prebiotics that would increase the concentration of organism A. This probiotic or probiotic may ⁇ be the exact organism A or another organism what would support the grown of organism A. The dose given would consider relative abundance of organisms in the individual, performance characteristics of the prebiotic/probiotic such as growth rate, compatibility, receptors or receptor density, genes, or expression patterns, or metabolomic products.
  • Custom tailored probiotics may not be in equal amounts but are formulated based on relative abundance detected from the individual gut/fecal sample. These formulations are geared to modulate the microbiome to a healthy status.
  • the healthy status of a microbiome is determined by the use of existing aggregate private and public databases such as metaHITTM, Human Microbiome ProjectTM, American Gut ProjectTM, and the like.
  • the healthy status may also be determined individually when a person has no known issues and is in good health, from a blood biomarker checkup perspective, and then has their full microbiome profile completed.
  • microbiome profiles can be aggregated into groups that are then assigned a barcode for rapid bioinformatic assignment. Groups can be created by single or multiple phenotypic, diagnostic, or demographic information related to the individual from which the sample was collected from. A unique group can be determined from another group by using statistical models such as linear distance calculations, diversity values, classifiers such as C4.5 decision tree, or principal component analysis an comparing to an aggregate known population such as "normals" defined by the Human Microbiome Project or American Gut Project.
  • the present invention may be used to screen the gut microbiome of a given subject and then custom tailor a probiotic regimen to the given subject based on the subject's gut microbiome.
  • the present invention may be used to restore a subject's gut flora and/or fauna to homeostasis after an event that has caused a shift in the subject's microbiota from balanced microbiome to one that is causing or may be causing negative side effects, disorders, and/or disease.
  • Health conditions can include but is not limited to various conditions, from acne and allergies, through gastrointestinal ailments, obesity and cancer.
  • One example of such a dysbiosis is in the case of the onset of obesity.
  • Several strains of microbes in the guts of subjects have been shown to be associated with obesity or weight management issues suffered by the subjects. See, e.g., Ley, et al. (2005) PNAS USA 102: 11070-11075.
  • a ratio of a first given microbe to a second given microbe in the gut of a subject is determined using the methods described herein and then if the ratio is undesired or abnormal, the subject is administered a treatment to modify the ratio to be a desired ratio.
  • the amount of a first given microbe in a gut of a subject relative to the total amount of all the microbes in the gut of the subject is determined using the methods described herein and then if the relative amount of the first given microbe is undesired or abnormal, the subject is administered a treatment to modify the amount to be a desired amount.
  • Such treatments include administering to the subject: a probiotic containing one or more microbes whose amounts are desired to be increased in the gut of the subject, an antimicrobial agent, e.g., an antibiotic, an antifungal, an antiviral, etc., to kill or slow the growth of a microbe or microbes whose amounts are desired to be decreased in the gut of the subject, a diet and/or a dietary supplement that supports the growth or maintenance of a healthy gut microbiome, e.g., a prebiotic, magnesium, fish oil, L-glutamine, vitamin D, etc., and the like.
  • an antimicrobial agent e.g., an antibiotic, an antifungal, an antiviral, etc.
  • the gut microbiota of obese subjects are enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii. Therefore, after determining the amounts of Lactobacillus reuteri, Bifidobacterium animalis, and Methanobrevibacter smithii in the gut of a subject using the methods described herein and finding that the amounts are typical or indicative of obesity-associated gut microbiota, the subject may be administered a probiotic containing Bifidobacterium animalis and Methanobrevibacter smithii and relatively little to no amount of Lactobacillus reuteri.
  • Scoring of the microbiome signature overall uses a similar decision tree, algorithm, artificial intelligence, script, or logic tree as represented in table 1. This system would enable a score that helps a user understand how healthy their gut microbiome is and if they need to take action on a few or many challenges found. Challenges can include but not limited to, identification of known pathogenic organisms, count and identification of opportunistic pathogens, latent organisms known to cause pathogenic affects when given opportunity, lack of support for good microbial environment but their composition or lack of key strains, overall diversity and count of unique organisms found in top 10 and or organisms with greater than 0.1% prevalence.
  • Table 1 An example of a scoring and probiotic formula algorithm is included in Table 1 below.
  • Table 1 can be represented as decision tree, algorithm, artificial intelligence, script, or logic tree. The function of such decision tree, algorithm, artificial intelligence, script, or logic tree would be output a score of wellness of the individual microbiome as related to probiotics detected and to provide formulation and dosing recommendations for probiotic usage.
  • Table 1 Example decision table for probiotic scoring and formulation. Includes the utilizatiton of a probiotic strain database, metagenomic analysis database, and liteature curati on database.
  • Table 2 Potential categories from which to create groups.
  • the term "subject” includes humans and non-human animals.
  • the term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals.
  • a and/or B means “A, B, or both A and B” and "A, B, C, and/or D” means “A, B, C, D, or a combination thereof and said "combination thereof means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).
  • a single member subset e.g., A or B or C or D
  • a two-member subset e.g., A and B; A and C; etc.
  • a three-member subset e.g., A, B, and C; or A, B, and D; etc.
  • all four members e.g., A
  • sample and “biological sample” refer to any sample suitable for the methods provided by the present invention.
  • a sample of cells can be any sample, including, for example, gut or fecal sample obtained by non-invasive or invasive techniques such as biopsy of a subject.
  • sample refers to any preparation derived from fecal matter or gut tissue of a subject.
  • a sample of cells obtained using the non-invasive method described herein can be used to isolate nucleic acid molecules or proteins for the methods of the present invention.
  • analysis can be of any nucleic acid, including DNA, RNA, cDNA, miRNA, mtDNA, single or double-stranded.
  • This nucleic acid can be of any length, as short as oligos of about 5 bp to as long a megabase or even longer.
  • nucleic acid molecule means DNA, RNA, single-stranded, double-stranded or triple stranded and any chemical modifications thereof. Virtually any modification of the nucleic acid is contemplated.
  • a "nucleic acid molecule” can be of almost any length, from 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in length, up to a full-length chromosomal DNA molecule.
  • the nucleic acid isolated from a sample is typically RNA.
  • a single-stranded nucleic acid molecule is "complementary" to another single- stranded nucleic acid molecule when it can base-pair (hybridize) with all or a portion of the other nucleic acid molecule to form a double helix (double-stranded nucleic acid molecule), based on the ability of guanine (G) to base pair with cytosine (C) and adenine (A) to base pair with thymine (T) or uridine (U).
  • G guanine
  • C cytosine
  • A adenine
  • T thymine
  • U uridine
  • the nucleotide sequence 5'-TATAC-3' is complementary to the nucleotide sequence 5'-GTATA-3' .
  • hybridization refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing.
  • Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations.
  • suitably stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • hybridization under high stringency conditions could occur in about 50% formamide at about 37°C to 42°C.
  • Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C.
  • hybridization could occur under high stringency conditions at 42°C in 50% formamide, 5X SSPE, 0.3%) SDS, and 200 mg/ml sheared and denatured salmon sperm DNA.
  • Hybridization could occur under reduced stringency conditions as described above, but in 35% formamide at a reduced temperature of 35°C.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.
  • microbiome refers to microorganisms, including bacteria, viruses, and fungi, archaea, protozoa, amoeba, or helminths that inhabit the gut of the subject.
  • microbial, microbe, or microorganism refer to any microscopic organism including prokaryotes or eukaryotes, spores, bacterium, archeaebacterium, fungus, virus, or protist, unicellular or multicellular.
  • the present invention is described partly in terms of functional components and various processing steps. Such functional components and processing steps may be realized by any number of components, operations and techniques configured to perform the specified functions and achieve the various results.
  • the present invention may employ various biological samples, biomarkers, elements, materials, computers, data sources, storage systems and media, information gathering techniques and processes, data processing criteria, statistical analyses, regression analyses and the like, which may carry out a variety of functions.
  • the invention is described in the medical diagnosis context, the present invention may be practiced in conjunction with any number of applications, environments and data analyses; the systems described herein are merely exemplary applications for the invention.
  • Methods for data analysis according to various aspects of the present invention may be implemented in any suitable manner, for example using a computer program operating on the computer system.
  • An exemplary analysis system may be implemented in conjunction with a computer system, for example a conventional computer system comprising a processor and a random access memory, such as a remotely-accessible application server, network server, personal computer or workstation.
  • the computer system also suitably includes additional memory devices or information storage systems, such as a mass storage system and a user interface, for example a conventional monitor, keyboard and tracking device.
  • the computer system may, however, comprise any suitable computer system and associated equipment and may be configured in any suitable manner.
  • the computer system comprises a stand-alone system.
  • the computer system is part of a network of computers including a server and a database.
  • the software required for receiving, processing, and analyzing genetic information may be implemented in a single device or implemented in a plurality of devices.
  • the software may be accessible via a network such that storage and processing of information takes place remotely with respect to users.
  • the analysis system according to various aspects of the present invention and its various elements provide functions and operations to facilitate microbiome analysis, such as data gathering, processing, analysis, reporting and/or diagnosis.
  • the present analysis system maintains information relating to microbiomes and samples and facilitates analysis and/or diagnosis,
  • the computer system executes the computer program, which may receive, store, search, analyze, and report information relating to the microbiome.
  • the computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate a models and/or predictions.
  • the analysis system may also provide various additional modules and/or individual functions.
  • the analysis system may also include a reporting function, for example to provide information relating to the processing and analysis functions.
  • the analysis system may also provide various administrative and management functions, such as controlling access and performing other administrative functions.

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